Diversity of Variable Number Tandem Repeat Loci in Shigella Species Isolated from Pediatric Patients.

Multilocus variable number tandem repeat (VNTR) analysis (MLVA) is a new typing method with several advantages compared to other methods. Dissemination of Shigella is highly significant in developing countries. Whilst Shigella is becoming increasingly important as an etiologic agent of pediatric shigellosis in Iran, little is known about the genetic diversity of the local strains. Therefore, the aim of this study was to describe the genetic diversity of Shigella species isolated from pediatric patients in Tehran, Iran. A total of 53 Shigella isolates were obtained from 1070 patients with diarrhea (less than 12 years of age). All isolates were identified by routine biochemical and serological tests. The confirmed Shigella isolates were further serogrouped (by the slide agglutination) using slide agglutination method. MLVA assay with the seven loci resolved 53 Shigella isolates into 36 different genotypes. Almost all the isolates were classified into five clonal complexes. Furthermore, our MLVA assay could effectively distinguish the four Shigella species. This study has provided valuable insights into the genetic heterogeneity of Shigella species in Tehran, Iran. Our findings can be helpful for further epidemiological surveillance of Shigella species in this country in the future.

Although PFGE is still a golden standard for genotyping and source tracking of food-borne pathogens, it is laborious, expensive, often difficult to interpret, and requires rigorous standardization.
Furthermore, it needs experienced personnel in order to achieve reliable, consistent, and reproducible results. By contrast, MLVA is a PCRbased genotyping method which is rapid, relatively cheap and easy to perform. This method targets multiple VNTR loci and relies on the detection of different copy numbers inside each locus (18).
Dissemination of Shigella is highly significant in developing countries. Whilst Shigella is becoming increasingly important as an etiologic agent of pediatric shigellosis in Iran (2,3,19), little is known about the genetic diversity of the local strains. To the best of our knowledge, this is the first study on genotype of Shigella by MLVA in Iran. Therefore, the aim of this study was to describe the genetic diversity of Shigella species isolated from pediatric patients in Tehran, Iran.

Bacterial strains
In this study, from June 2008 to October 2010, a total of 53 Shigella isolates were obtained from 1070 patients with diarrhea (less than 12 years of age) in Tehran, Iran. All of the isolates were identified by routine biochemical and serological tests. The confirmed Shigella isolates were further serogrouped by the slide agglutination test (Mast Diagnostic, Merseyside, UK). The verified isolates were preserved at -70°C in tripticase soy broth (TSB) with 25% (v/v) glycerol for further analysis (3). All ethical issues were considered. Life, health, dignity, integrity, right to self-determination, privacy, and confidentiality of personal information of research subjects were protected in this study.

DNA extraction
Each Shigella isolate was plated on nutrient agar and incubated overnight at 37 °C. A single colony was removed from the plate, suspended in 200 µl of sterile deionized water and boiled for 15 min. After centrifugation at 8,000x g for 6 min, the supernatant was transferred into a new tube for subsequent PCR analysis.

MLVA assay
The VNTR loci selected along with the primers used for MLVA genotyping were those of Gorge et al., which were seven loci; ms06, ms07, ms09, ms11, ms21, ms23, and ms32 (20). The list of primers used are shown in Table 1  S.flexneri Sf2457T (serotype 2a) was also included as an outgroup.

Results
Of the 53 Shigella isolates, S. sonnei (50.9%,     (27). Generally, these studies showed that MLVA has the potential to replace other genotyping methods as a standard method for Shigella typing. However, lack of standardization of the methodology and interpretive criteria is problematic and hinders comparison of data between laboratories (9).
On the other hand, it is important to note that most of these mentioned MLVA schemes require a high precision of DNA length measurement, for instance, by microcapillary electrophoresis and fluorescent markers because the selected VNTRs have very short repeat units. Moreover, developing countries have limited accessibility to such equipment and their expenditures would be a major obstacle for many laboratories (20,29). In this study, we exploited VNTR markers which can be easily analyzed by eye on agarose gels. Hence, this assay can be performed in a laboratory equipped with simple equipment.
The present study provided valuable insights into the genetic heterogeneity of Shigella species in Tehran. We hope that our findings can be helpful for further epidemiological surveillance of Shigella species in our country in the future. Finally, this study showed that MLVA is a promising typing technique for epidemiological studies of Shigella species due to low cost, the speed of analysis, and the ability to produce numerical data which can be easily shared among laboratories.